The Broad Goals of this proposal are: i) to define the basic machinery of secretory transport in African trypanosomes, and ii) to investigate aspects of secretory trafficking that are unique to these parasites. African trypanosomes cause disease in both humans (sleeping sickness) and cattle (nagana). They avoid the host immune response be repeatedly changing the expression of VSGs (variant surface glycoprotein); the major glycosylphosphatidyl-inositol (GPI) anchored antigens. Consequently, the secretory trafficking of essential surface proteins, such as VSG, is critical to the success of trypanosomes as parasites. The interrelated goals of this proposal are contained in three Specific Aims. First, trypanosomal homologs of two related ATPases that are essential for the fusion of transport vesicles with target membranes throughout the secretory pathway will be biochemically characterized. These ATPases are VCP (valosin-containing protein) and NSF (NEM-sensitive fusion protein). Dominant negative ATPase mutants of these proteins will be engineered and expressed in transgenic T. brucei, resulting in accumulation of free transport vesicles and elevated association of the mutant ATPases with the membrane receptors that provide specificity to vesicle docking and fusion. This will allow direct analysis of the composition and cargo of secretory vesicles, and the identification of specific membrane markers for secretory compartments in trypanosomes. Second, the role of GPI anchors in forward secretory transport will be investigated. Prevention of GPI addition dramatically reduces the rate of VSG transport in trypanosomes, leading to accumulation of VSG in the endoplasmic reticulum. The sufficiency of GPI anchors to mediate secretory transport will be assessed by engineering GPI addition to other secretory cargo proteins. Acceleration of the rate of transport of these chimeric reporters will establish that GPI anchors act as positive forward transport signals in the earliest part of the trypanosomal secretory pathway. Experiments will then be designed to ask if GPI-dependent transport is best explained by a receptor-mediated or a lipid microdomain model. Third, a metalloprotease that releases recombinant VSG from the surface of transgenic procyclic trypanosomes will be biochemically characterized. This release mimics the turnover of VSG from bloodstream trypanosomes during differentiation; thus this enzyme may play an important role in the normal metabolism of VSG. To address this issue the expression of the protease during differentiation will be disrupted and the resulting effects on VSG release, and the entire differentiation process, will be determined. Overall, these studies will provide a greater understanding of essential secretory processes in trypanosomes and will illuminate the role of secretion in both the pathogenesis of trypanosomiasis and the reorganization of trypanosomal cell surface architecture during life cycle differentiation.